In recent years, genetic information of many organisms including human genome has been decoded. Under the circumstances, functional analysis of proteins corresponding to such genetic information and creation of genomic medicine have been attracting attention as postgenomic studies. Application and utilization of proteins corresponding to such genetic information for pharmaceutical products and the like requires easy syntheses of extensive kinds of proteins in a short time.
At present, expression systems using viable cells (hereinafter sometimes to be referred to as “cell-system”) of yeast, insect cell and the like by gene recombination technique have been widely used as the production methods of proteins. However, many proteins are difficult to express. For example, viable cells show a propensity toward elimination of exogenous proteins for their functional retention, and expression of cytotoxic proteins in viable cells prevents cell growth.
As a production method of a protein that does not use a cell-system, a cell-free protein synthesis has been known, which includes adding a substrate, enzyme and the like to a cell rupture and extract solution and the like to provide a wide choice of genetic information translation systems of organism in test tubes, and reconstructing a synthetic system capable of linking the necessary number of residues in a desired order of amino acids using an mRNA encoding the objective protein. Such a cell-free protein synthesis is not easily limited unlike the above-mentioned cell-system protein synthesis, and proteins can be synthesized without killing the organism. In addition, because the production of protein does not accompany operations such as cultivation and the like, a protein can be synthesized in a short time as compared to cell-systems. Moreover, inasmuch as the cell-free protein synthesis also affords a large-scale production of proteins consisting of the amino acid sequence that the organism does not use, it is expected to be a promising expression method. As such cell-free protein synthesis, for example, methods using an extract solution of wheat germ and that of Escherichia coli have been known.
In a cell-free protein synthesis using an extract solution of wheat germ, however, the extraction process for the extract solution is generally extremely complicated.
As one example of the preparation method of an extract solution of wheat germ, JP-A-2000-236896 describes the following steps. Wheat seeds are added in a mill, ruptured and a crude germ fraction is obtained using a sieve. By flotation with a mixture of carbon tetrachloride and cyclohexane (carbon tetrachloride:cyclohexane=2.5:1), germinative embryo is recovered from the floating fractions and the organic solvent is removed by drying at room temperature. The impurities contained in the embryo fraction, such as seed coat and the like, are removed by adsorption using a static electricity charged body. Then, to completely remove a wheat albumen component from this sample, it is suspended in a 0.5% solution of NP40, a nonionic detergent, and repeatedly washed with an ultrasonic cleaner until the washing does not become cloudy. Ultrasonic cleaning is done once again in the presence of distilled water to purify the wheat germ.
The cell-free protein synthesis using an extract solution of wheat germ in this way requires complicated preparation of an extract solution, inconveniently demanding long hours and much labor.
A cell-free protein synthesis using an extract solution of Escherichia coli fails in glycosylation to a protein, because Escherichia coli is a procaryote, and cannot synthesize glycoprotein. The sugar chain added to a protein by the above-mentioned glycosylation is considered to function as a function regulating factor of a protein itself or a protective and stabilizing factor of protein, in the form of a signal or ligand involved in the recognition and adhesion between substances or between cells. For the analysis of in vivo function of a protein to be glycosylated, a glycosylated protein (glycoprotein) should be obtained. Thus, there is a demand for a cell-free protein synthesis that permits glycosylation after translation into a protein.